Modular, cement-free, customized headpost and connector-chamber implants for macaques.

BACKGROUND: Neurophysiological studies with awake macaques typically require chronic cranial implants. Headpost and connector-chamber implants are used to allow head stabilization and to house connectors of chronically implanted electrodes, respectively. NEW METHOD: We present long-lasting, modular, cement-free headpost implants made of titanium that consist of two pieces: a baseplate and a top part. The baseplate is implanted first, covered by muscle and skin and allowed to heal and osseointegrate for several weeks to months. The percutaneous part is added in a second, brief surgery. Using a punch tool, a perfectly round skin cut is achieved providing a tight fit around the implant without any sutures. We describe the design, planning and production of manually bent and CNC-milled baseplates. We also developed a remote headposting technique that increases handling safety. Finally, we present a modular, footless connector chamber that is implanted in a similar two-step approach and achieves a minimized footprint on the skull. RESULTS: Twelve adult male macaques were successfully implanted with a headpost and one with the connector chamber. To date, we report no implant failure, great headpost stability and implant condition, in four cases even more than 9 years post-implantation. COMPARISON WITH EXISTING METHODS: The methods presented here build on several related previous methods and provide additional refinements to further increase implant longevity and handling safety. CONCLUSIONS: Optimized implants can remain stable and healthy for at least 9 years and thereby exceed the typical experiment durations. This minimizes implant-related complications and corrective surgeries and thereby significantly improves animal welfare.

Fast-field-cycling NMR at very low magnetic fields: water molecular dynamic biomarkers of glioma cell invasion and migration.

Our objective was to study NMR relaxometry of glioma invasion/migration at very low field (<2 mT) by fast-field-cycling NMR (FFC-NMR) and to decipher the pathophysiological processes of glioma that are responsible for relaxation changes in order to open a new diagnostic method that can be extended to imaging. The phenotypes of two new glioma mouse models, Glio6 and Glio96, were characterized by T2w -MRI, HE histology, Ki-67 immunohistochemistry (IHC) and CXCR4 RT-qPCR, and were compared with the U87 model. R1 dispersions of glioma tissues were acquired at low field (0.1 mT-0.8 T) ex vivo and were fitted with Lorentzian and power-law models to extract FFC biomarkers related to the molecular dynamics of water. In order to decipher relaxation changes, three main invasion/migration pathophysiological processes were studied: hypoxia, H2 O2 function and the water-channel aquaporin-4 (AQP4). Glio6 and Glio96 were characterized with invasion/migration phenotype and U87 with high cell proliferation as a solid glioma. At very low field, invasion/migration versus proliferation was characterized by a decrease in the relaxation-rate constant (ΔR1 ≈ -32% at 0.1 mT) and correlation time (≈-40%). These decreases corroborated the AQP4-IHC overexpression (Glio6/Glio96: +92%/+46%), suggesting rapid transcytolemmal water exchange, which was confirmed by the intracellular water-lifetime τIN decrease (ΔτIN ≈ -30%). In functional experiments, AQP4 expression, τIN and the relaxation-rate constant at very low field were all found to be sensitive to hypoxia and to H2 O2 stimuli. At very low field the role of water exchanges in relaxation modulation was confirmed, and for the first time it was linked to the glioma invasion/migration and to its main pathophysiological processes: hypoxia, H2 O2 redox signaling and AQP4 expression. The method appears appropriate to evaluate the effect of drugs that can target these pathophysiological mechanisms. Finally, FFC-NMR operating at low field is demonstrated to be sensitive to invasion glioma phenotype and can be straightforwardly extended to FFC-MRI as a new cancer invasion imaging method in the clinic.

GluA4-Targeted AAV Vectors Deliver Genes Selectively to Interneurons while Relying on the AAV Receptor for Entry.

Selective gene delivery into subtypes of interneurons remains an important challenge in vector development. Adeno-associated virus (AAV) vector particles are especially promising for intracerebral injections. For cell entry, AAV2 particles are supposed to attach to heparan-sulfate proteoglycans (HSPGs) followed by endocytosis via the AAV receptor (AAVR). Here, we assessed engineered AAV particles deficient in HSPG attachment but competent in recognizing the glutamate receptor 4 (GluA4, also known as GluRD or GRIA4) through a displayed GluA4-specific DARPin (designed ankyrin repeat protein). When injected into the mouse brain, histological evaluation revealed that in various regions, more than 90% of the transduced cells were interneurons, mainly of the parvalbumin-positive subtype. Although part of the selectivity was mediated by the DARPin, the chosen spleen focus-forming virus (SFFV) promoter had contributed as well. Further analysis revealed that the DARPin mediated selective attachment to GluA4-positive cells, whereas gene delivery required expression of AAVR. Our data suggest that cell selectivity of AAV particles can be modified rationally and efficiently through DARPins, but expression of the AAV entry receptor remains essential.

In vivo quantification of magnetically labelled cells by MRI relaxometry.

Cellular MRI, which visualizes magnetically labelled cells (cells*), is an active research field for in vivo cell therapy and tracking. The simultaneous relaxation rate measurements (R2 *, R2 , R1 ) are the basis of a quantitative cellular MRI method proposed here. U937 cells were labelled with Molday ION Rhodamine B, a bi-functional superparamagnetic and fluorescent nanoparticle (U937*). U937* viability and proliferation were not affected in vitro. In vitro relaxometry was performed in a cell concentration range of [2.5 × 104 -108 ] cells/mL. These measurements show the existence of complementary cell concentration intervals where these rates vary linearly. The juxtaposition of these intervals delineates a wide cell concentration range over which one of the relaxation rates in a voxel of an in vivo image can be converted into an absolute cell concentration. The linear regime was found at high concentrations for R1 in the range of [106 - 2 × 108 ] cells/mL, at intermediate concentrations for R2 in [2.5 × 105 - 5 × 107 ] cells/mL and at low concentrations for R2 * in [8 × 104 - 5 × 106 ] cells/mL. In vivo relaxometry was performed in a longitudinal study, with labelled U937 cells injected into a U87 glioma mouse model. Using in vitro data, maps of in vivo U937* concentrations were obtained by converting one of the in vivo relaxation rates to cell concentration maps. MRI results were compared with the corresponding optical images of the same brains, showing the usefulness of our method to accurately follow therapeutic cell biodistribution in a longitudinal study. Results also demonstrate that the method quantifies a large range of magnetically labelled cells*. Copyright © 2016 John Wiley & Sons, Ltd.

Beta oscillation dynamics in extrastriate cortex after removal of primary visual cortex.

The local field potential (LFP) in visual cortex is typically characterized by the following spectral pattern: before the onset of a visual stimulus, low-frequency oscillations (beta, 12-20 Hz) dominate, whereas during the presentation of a stimulus these oscillations diminish and are replaced by fluctuations at higher frequencies (gamma, >30 Hz). The origin of beta oscillations in vivo remains unclear, as is the basis of their suppression during visual stimulation. Here we investigate the contribution of ascending input from primary visual cortex (V1) to beta oscillation dynamics in extrastriate visual area V4 of behaving monkeys. We recorded LFP activity in V4 before and after resecting a portion of V1. After the surgery, the visually induced gamma LFP activity in the lesion projection zone of V4 was markedly reduced, consistent with previously reported spiking responses (Schmid et al., 2013). In the beta LFP range, the lesion had minimal effect on the normal pattern of spontaneous oscillations. However, the lesion led to a surprising and permanent reversal of the normal beta suppression during visual stimulation, with visual stimuli eliciting beta magnitude increases up to 50%, particularly in response to moving stimuli. This reversed beta activity pattern was specific to stimulus locations affected by the V1 lesion. Our results shed light on the mechanisms of beta activity in extrastriate visual cortex: The preserved spontaneous oscillations point to a generation mechanism independent of the geniculostriate pathway, whereas the positive beta responses support the contribution of visual information to V4 via direct thalamo-extrastriate projections.

Cerebral blood volume quantification in a C6 tumor model using gadolinium per (3,6-anhydro) alpha-cyclodextrin as a new magnetic resonance imaging preclinical contrast agent.

In magnetic resonance imaging (MRI), cerebral blood volume (CBV) quantification is dependent on the MRI sequence and on the properties of the contrast agents (CAs). By using the rapid steady-state T(1) method, we show the potential of gadolinium per (3,6-anhydro) alpha-cyclodextrin (Gd-ACX), a new MRI paramagnetic CA (inclusion complex of Gd(3+) with per (3,6-anhydro)-alpha-cyclodextrin), for the CBV quantification in the presence of blood-brain barrier lesions. After biocompatibility and relaxivity experiments, in vivo experiments on rats were performed on a C6 tumor model with 0.05 mmol Gd-ACX/kg (<1/10 of the median lethal dose) injected at a 25 mmol/L concentration, inducing neither nephrotoxicity nor hemolysis. On T(1)-weighted images, a signal enhancement of 170% appeared in vessels after injection, but not in the tumor (during the 1 h of observation), in contrast to the 90% signal enhancement obtained with Gd-DOTA (a clinical MRI CA) injected at a T(1) isoefficient dose. This result shows the absence of Gd-ACX extravasation into the tumor tissue and its confinement to the vascular space. Fractional CBV values were found similar to Gd-ACX and Gd-DOTA in healthy brain tissue and in the contralateral hemisphere of tumor-bearing rats, whereas only Gd-ACX was appropriate for CBV quantification in tumor regions.

Inability to directly detect magnetic field changes associated with neuronal activity.

The ability to directly detect neuronal magnetic fields by MRI would help investigators achieve the "holy grail" of neuroimaging, namely both high spatial and temporal resolution. Both positive and negative findings have been reported in the literature, with no clear consensus as to the feasibility of direct detection. The aim of this study was to replicate one of the most promising published in vivo results. A second aim was to investigate the use of steady-state visual evoked potentials (ssVEPs), which give a large evoked response and offer a well-controlled approach because the frequency of the neuronal response can be dictated by the experimenter. For both studies we used a general linear model (GLM) that included regressors for both the expected blood oxygen level-dependent (BOLD) signal and the magnetic source (MS) signal. The results showed no activity that could be attributed to the neuromagnetic signals in either study, and no frequency component corresponding to the frequency of the ssVEPs. This study demonstrates that for the particular stimuli and hardware used, the sensitivity of the magnitude MRI signal to detect evoked neuronal currents is too low to be of practical use.

Assessing neuronal coherence with single-unit, multi-unit, and local field potentials.

The purpose of this study was to obtain a better understanding of neuronal responses to correlated input, in particular focusing on the aspect of synchronization of neuronal activity. The first aim was to obtain an analytical expression for the coherence between the output spike train and correlated input and for the coherence between output spike trains of neurons with correlated input. For Poisson neurons, we could derive that the peak of the coherence between the correlated input and multi-unit activity increases proportionally with the square root of the number of neurons in the multi-unit recording. The coherence between two typical multi-unit recordings (2 to 10 single units) with partially correlated input increases proportionally with the number of units in the multi-unit recordings. The second aim of this study was to investigate to what extent the amplitude and signal-to-noise ratio of the coherence between input and output varied for single-unit versus multi-unit activity and how they are affected by the duration of the recording. The same problem was addressed for the coherence between two single-unit spike series and between two multi-unit spike series. The analytical results for the Poisson neuron and numerical simulations for the conductance-based leaky integrate-and-fire neuron and for the conductance-based Hodgkin-Huxley neuron show that the expectation value of the coherence function does not increase for a longer duration of the recording. The only effect of a longer duration of the spike recording is a reduction of the noise in the coherence function. The results of analytical derivations and computer simulations for model neurons show that the coherence for multi-unit activity is larger than that for single-unit activity. This is in agreement with the results of experimental data obtained from monkey visual cortex (V4). Finally, we show that multitaper techniques greatly contribute to a more accurate estimate of the coherence by reducing the bias and variance in the coherence estimate.